A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

Paul J. Hergenrother,Philip D. Reynolds,Nicole M. Kretzer,Elizabeth M. Wilson,Chengjian Mao,David J. Shapiro,Irene O. Aninye,Milu T. Cherian,Rachel Schiff,Steven K. Nordeen

doi:10.1074/jbc.m110.183723

Abstract

The mechanisms responsible for 17β-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

Highlights

The mechanisms responsible for 17␤-estradiol (E2)-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor ␣ (ER␣) antagonists are not fully understood
We describe a new tool for dissecting ER␣ action in breast cancer, p-fluoro-4-(1,2,3,6,tetrahydro1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor ␣ that does not compete with estrogen for binding to ER␣
Reporter Gene Assays—Reporter gene assays were performed to compare the ability of about 200 compounds structurally related to TPBM [19] to inhibit estrogen-dependent transcription in T47D-KBluc breast cancer cells stably transfected to express an (ERE)3-Luc reporter [24]

Summary

The abbreviations used are

ER␣, estrogen receptor ␣; E2, 17␤-estradiol; TPSF, butyrophenone, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo6-thionpurin-8-ylthio); TPBM, 8-benzylsulfanylmethyl-1,3-dimethyl-3,7dihydropurine-2,6-dione; OHT, 4-hydroxytamoxifen; Luc, luciferase; MMTV, mouse mammary tumor virus; PSA, prostate-specific antigen; AR, androgen receptor; GR, glucocorticoid receptor; ERE, estrogen response element; flcERE, fluorescein-labeled consensus ERE; AIB1, amplified in breast cancer 1; Dox, doxycycline; qRT-PCR, quantitative reverse transcriptase-PCR; CD, charcoal dextran; PI-9, proteinase inhibitor 9; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PR, progesterone receptor; MEM, minimum Eagle’s medium; CS, calf serum. A Novel Inhibitor of ER␣ Action in Breast Cancer Cells tagonists that act outside the ligand binding pocket of ER␣. We recently described an in vitro high throughput screening strategy to identify small molecule inhibitors of ER␣ binding to DNA. We identified 8-benzylsulfanylmethyl-1,3-dimethyl3,7-dihydropurine-2,6-dione (TPBM) as a small molecule inhibitor of ER␣ binding to ERE DNA [19]. Using a cell-based screen, we evaluated ϳ200 small molecules structurally related to TPBM and identified butyrophenone, p-fluoro-4(1,2,3,6, -tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF) as a novel inhibitor of ER␣ Ͼ15-fold more potent than TPBM. TPBM inhibits in vitro binding of E2-ER␣ to a labeled ERE, TPSF does not. We show that TPSF inhibits anchorage-dependent and anchorage-independent growth of tamoxifen-sensitive and tamoxifen-resistant ER␣-containing breast cancer cells and demonstrate that TPSF enhances proteasome-dependent degradation of ER␣

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION